Vascularization of the cornea is a vision-threatening complication. The molecular mechanisms underlying corneal avascularity are still not well understood and comparatively little is known about the development of corneal avascularity. We propose to study the molecular regulation of migratory blood vessel precursor cells (angioblasts) during development of corneal avascularity. Previously, we showed that periocular angioblasts express Neuropilin1 (Nrp1) a dual receptor for the angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic guidance molecule Semaphorin3A (Sema3A). Our ongoing studies show that periocular angioblasts also express PlexinD1 a receptor for anti-angiogenic Sema3E during cornea development. We also show that the anti-angiogenic cytokine CXCL14 is strongly expressed by stromal keratocytes shortly after differentiation. Based on these observations we hypothesize that a tightly regulated balance between pro- and anti-angiogenic factors controls angioblast migration and vascular patterning during development of corneal avascularity. To test this hypothesis, we will take advantage of mouse genetics and the ease of manipulating avian eyes to explore the function of inhibitory genes associated with vasculogenesis during corneal development. We will examine the behavior of migratory angioblasts during eye development to determine where and when they segregate from other migratory cells that give rise to the cornea stroma and endothelium. We will determine the spatiotemporal expression of guidance genes and their receptors during cornea development by qPCR, in situ hybridization, and immunohistochemistry. We will perform loss- and gain-of-function experiments in Tie1:H2B-eYFP transgenic quail embryos with fluorescent blood vessels to elucidate the function of inhibitory molecules and their receptors during periocular angioblast migration. Knockout mice lacking the function of each inhibitory gene or receptor will be examined in detail for defects in angioblast migration and vascularization of the cornea. In vivo experiments will be coupled with in vitro migration assays to directly test the effect of inhibitory molecules on periocular angioblast migration in isolation from other ocular tissues. The following Specific Aims will test our hypothesis: 1. Determine the role of Nrp1 and its ligands VEGF and Sema3A in angioblast migration during development of corneal avascularity. 2. Identify the role of PlexinD1 during development of corneal avascularity. 3. Determine the functional significance of CXCL14 expression during cornea development. Successful completion of our proposed study will provide insight into the mechanisms that regulate angioblast migration and vasculogenesis leading to development of an avascular cornea. Similar mechanisms may inhibit neovascularization of the normal adult cornea and thus pave the way for discovery of potential therapeutic anti-angiogenesis agents for treating vascularized adult corneas and targeting angiogenesis due to cancer. PUBLIC HEALTH RELEVANCE: The transparent cornea is a highly innervated but remains avascular. Inappropriate vascularization of the cornea can result in loss of visual acuity or blindness. Angioblasts and endothelial cells express receptors for attractive and repulsive signals that guide their migration and vascularization of specific embryonic tissues. Angioblasts express receptors for several inhibitory molecules at a critical time as they migrate adjacent to the developing cornea. We propose to determine the role of inhibitory guidance molecules during the selective avoidance of migratory periocular angioblasts but not neural crest cells of the developing cornea, which results in its avascularity.